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FACE™ NFκB p65 Profiler

in-cell Western analysis for phospho NFκB p65 (S468 and S536)

Fast Activated Cell-based ELISA (FACE™) Kits provide a simple, sensitive method for detecting protein phosphorylation directly in the cell, without making extracts or performing electrophoresis and membrane blotting. These 96-well, high-throughput assays are available in both colorimetric and chemiluminescent formats for over 20 different targets (see list at right). For complete details, click the FACE™ Method tab below.

FACE NFκB p65 Profiler Kits provide 96 rxns each of 3 antibodies that enable you to monitor and compare the levels of NFκB p65 phosphorylated at two different sites, as well as total NFκB p65. The phospho-NFκB p65 (S468) antibody specifically recognizes NFκB p65 when phosphorylated at Ser468, while phospho-NFκB p65 (S536) antibody specifically recognizes NFκB p65 that is phosphorylated at Ser536. The total-NFκB p65 antibody recognizes NFκB p65 regardless of its phosphorylation state. Click the NFκB p65 Info tab below for data and more information.

 
Name Format Cat No. 価格 (税抜)  
FACE™ NFκB p65 Profiler 3 x 96 rxns 48300 ¥121,000 Buy Now
FACE™ NFκB p65 Profiler Chemi 3 x 96 rxns 48400 ¥131,000 Buy Now
Measurement of phosphorylated NFkB p65 (S468 and S536) and total NFkB p65 using the FACE NFkB p65 Profiler Cell Based ELISA (In Cell Western) method.
 
Figure 1: Measurement of phosphorylated and total NFκB p65.

HeLa cells were cultured in 96-well plates and serum-starved for 16 hours. Cells were then treated with 20 ng/ml TNF-α and 50 nM Calyculin A for 15 minutes and fixed. Total and phospho NFκB p65 were each assayed in triplicate using the phospho and total NFκB p65 antibodies included in the FACE NFκB p65 Profiler Kit. Data was plotted after correction for cell number (performed through use of Crystal Violet).

Antibody Specificities

The phospho-NFκB p65 (S468) was raised against a synthetic phospho-peptide corresponding to residues surrounding Serine 468 of human NFκB p65 and does not cross-react with other sites. The phospho-NFκB p65 (S536) was raised against a synthetic phospho-peptide corresponding to residues surrounding Serine 536 of human NFκB p65 and does not cross-react with other sites. The total-NFκB p65 antibody recognizes NFκB p65 proteins regardless of the phosphorylation state.

NFκB p65 Overview

The transcription factor NFκB (NF-kappa-B p65, RelA, nuclear factor κB) is a key component for the inducible expression of a wide variety of cellular and viral genes. NFκB is composed of a heterodimer of p65 and p50 subunits in most cell types and is sequestered in the cytoplasm by its inhibitory proteins, the IκBs. During the phosphorylation and degradation of IκBs, NFκB p65 is phosphorylated on multiple resides, each triggered by different stimuli but essential for maintaining NFκB transcriptional activation. Because of its role in regulating inflammatory and immune responses, high-throughput study methods for monitoring NFκB phosphorylation are in high demand.

FACE™ の使用法

96ウェルプレートで細胞を培養し, 目的の経路を誘導する刺激を与えます。刺激を与えた後, 細胞を固定することでリン酸化を含むタンパク質修飾が保持されます。ブロッキング反応の後, それぞれのウェルに目的タンパク質に特異的な一次抗体, 続いてHRP標識二次抗体を加えます。その後, 発色試薬を加えることにより, 比色定量または化学発光による定量を簡便に行うことができます (図1)。また, キット付属のCrystal Violetを用いて細胞数を補正することができます。それぞれのFACE Kitには目的タンパク質全体とリン酸化型を認識する一次抗体が含まれます。これにより, 細胞数と細胞中の目的タンパク質の総量に対してのリン酸化型タンパク質量を解析することができます。FACEはリン酸化型タンパク質を解析することのできるキットで, 定量的かつ再現性のある結果が得られます。

 
FACE flow chart
 
図1: FACE の操作法

Contents & Storage

Three 96-well plates for culturing cells, 96 rxns each of three primary antibodies (2 phospho-specific, 1 specific for native protein), HRP-conjugated secondary antibody, Quenching Solution, 1X Antibody Blocking Buffer, 1X Antibody Dilution Buffer, 10X PBS, 10% Triton X-100, 1% SDS Solution, Developing and Stop Solutions, and Crystal Violet Cell Quantification Solution. Storage conditions vary from room temperature to -20°C, see manual for details. All reagents are guaranteed stable for 6 months when stored properly.